Digital polymerase chain reaction (dpcr) platforms on next generation sequencer patterned flow cell technology

ABSTRACT

A method of detection of a target nucleic acid is provided. The method performs a digital polymerase chain reaction (dPCR) that extends the functionality of the next-generation sequencer on Patterned Flow Cell based technology. The method further includes partitioning a sample into the patterned flow cell, detecting the target nucleic acid, and counting the target nucleic acid.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority of U.S. Provisional Application No. 63/249,141 filed on Sep. 28, 2021 under 35 U.S.C. § 119(e), the entire contents of all of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Digital PCR (dPCR) is a quantitative PCR method that provides accurate, sensitive quantification of nucleic acids. dPCR incorporates traditional PCR reactions with fluorescence-based detection. The method assumes that sample partitioning will allow the application of Poisson statistic distribution resulting in 0 or 1 target per compartment. Once the sample is partitioned, the PCR amplification reactions amplify the sample in the chambers. After the PCR amplification completes, the presence or absence of fluorescence in the amplified reaction chamber is then used to calculate the absolute number of targets present in the initial sample. There are a few types of digital PCR in the field. For example, Droplet Digital PCR performs by segmenting samples using water-in-oil emulsions to create droplets from which their genetic material can be identified and quantified. Chip-based Digital PCR in which chip-based digital PCR is based on PCR reaction, load chips, PCR amplification, and fluorescence detection. The above approach can provide partition volumes ranging from femtoliters to almost microliter volumes.

dPCR is a practical method in diagnostic medicine and molecular biology research, including rare allele and CNV detection, accurate NGS library quantification, and high sensitivity for detecting targets in limited clinical samples.

Patterned flow cells are used in the next generation sequencer and have distinct wells across patterned flow cell surfaces at fixed locations. Each well possesses DNA probes to capture the qualified DNA for amplification during cluster generation. In addition, distinct wells provide partitioned compartments. The Illumina sequencers such as the HiSeq and NovoSeq use patterned flow cells to discriminate between much more packed DNA clusters.

SUMMARY OF THE INVENTION

The present invention provides a method of detecting target sequences that extends the functionality of the next-generation sequencer.

The invention provides a method of performing the digital PCR on the partitioned compartment of the patterned flow cell.

In certain aspects of the present invention, the method comprises the steps of (a) obtaining DNA molecules of samples; (b) ligating 5′ and 3′ adapter sequences to both ends of an individual DNA molecule strand; (c) attaching the 5′ or 3′ adapter modified individual DNA molecules to the reaction area of the patterned flow cell; (d) amplifying individual DNA molecules to form clusters on a reaction area of the patterned flow cell; (e) binding the fluorescent attached probe to individual DNA molecules interest in clusters; (f) generating the signal by a light exciting each of the clusters; and (g) imaging the patterned flow cell.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A illustrates the top view of the reaction area of the patterned flow cell, and FIG. 1B is the side view of the oligonucleotides in the reaction area of the patterned flow cell.

FIG. 2A illustrates the DNA molecules hybridizing the oligonucleotides on the patterned flow cell via the complementary sequence obeying the Poisson distribution. and FIG. 2B illustrates the amplification in the reaction area and replicating of the original DNA strand to form DNA clusters.

FIG. 3A illustrates the fluorescent-attached probe binds to the target DNA sequence in the reaction area, and FIG. 3B illustrates the imaging of the fluorescence emitted by exciting with light. White color circles represent a single target DNA sequence that binds to oligonucleotides originally. The striped circles represent multiple target DNA sequences that bind to oligonucleotides originally. The dark color circles represent no target DNA sequence binds to oligonucleotides originally.

DETAILED DESCRIPTION OF THE INVENTION

The disclosure provides a method for conducting a digital PCR assay on the next generation sequencer patterned flow cell.

In some embodiments, the specialized adapters are ligated to both ends of the DNA fragments of the sample in preparation for attaching to the patterned flow cell in the sequencing machine. In some embodiments, an ‘A’ base is added to the blunt ends of each DNA fragment, equipping them for ligation to the adapters. In some embodiments, the adapters possess a ‘T’ base overhang, supplying a complementary overhang for ligating the adapter to the A-tailed fragmented DNA. The adapter-ligated DNA fragments can bind to the reaction area of the patterned flow cell through hybridization. In certain embodiments, the adapters modified DNA includes at least two “flow cell” binding sites, each corresponding to the oligonucleotides in the reaction area of the patterned flow cell. FIG. 1A illustrates an exemplary top view of the patterned flow cell. Component 1 is one of reaction area with oligonucleotide sequences on patterned flow cell.

In some embodiments, the shape of the reaction areas in a patterned flow cell can be well-like or pillar-like. The photo-polymerizable material and oligonucleotide can be coated on the well of the reaction area to form gel reaction areas. FIG. 1B illustrates an exemplary side view of the patterned flow cell. Component 2 is the oligonucleotide sequences cluster.

In some embodiments, the fragmented DNA is washed over the patterned flow cell. The adapter modified individual DNA molecules attached to the complementary oligonucleotides on the reaction area. FIG. 2A illustrates an adapter modified individual DNA molecule strand sequence 21 hybridized to the complementary oligonucleotides cluster with oligonucleotides 22 on patterned flow cell.

In some embodiments, the present invention provides for amplification of partitioned DNA fragments; for example, Illumina's “bridge amplification PCR” creates clusters by repeatedly bending each fragment such that its second adapter hybridizes to an oligonucleotide in its surroundings and uses it as a template to create the complementary sequence. This form of PCR makes colonies with amplicons in both orientations, leaving a cluster of identical, single-stranded DNA templates for each DNA fragment that was originally hybridized on the patterned flow cell. FIG. 2B illustrates the amplification of partitioned DNA fragments in cluster 23.

In some embodiments, the probe with fluorescent contains a complementary sequence for binding DNA fragments in the colonies. FIG. 3A illustrates the probe with a fluorophore 31 washing over the patterned flow cell. The probe binding is considered part of the detection that a sample molecule target was present. In FIG. 3A , the probes with fluorophore 32 hybridize the DNA molecules in the cluster of patterned flow cell. In some embodiments, the set of capture probes is prepared by a reference sequencing library that is substantially complementary to a part of or adjacent to a region of interest DNA fragments. In some embodiments, the signal strength is based on a number of DNA fragments hybridized with capture probes in the clusters. In some embodiments, the imaging system detects the capture probes with detectable molecules emitting distinguishable signals. For example, one probe may be labeled with a fluorophore. In some embodiments, the label probes can include a plurality of identical label probes, different types, or probes of the same kind but provide other detectable signals.

In some embodiments, the analysis utilizes fluorescent probes and light-based detection methods to identify the products of DNA fragments colonies. In a practical embodiment, the energy source may be selected from a fluorescence excitation source but is not limited. A fluorescence excitation source, as used herein, is any entity capable of making a source fluoresce or producing photonic emissions. FIG. 3B illustrates the emitting light from clusters upon the light excitation. Signal 33 is emitted fluorescence, signal 34 is no emitted fluorescence, and signal 35 is mixed emitted fluorescence.

In some embodiments, the nucleic acid targets analysis involves detecting signals from the detectable molecules and the images. In some embodiments, it may be desirable to further label the target nucleic acid molecule with a standard marker that compares information obtained from different targets. 

1. A method for performing digital PCR on next generation sequencer patterned flow cell comprising: (a). attaching the adapters to each end of the individual DNA molecules derived from biological samples; (b) distributing the individual DNA molecules on the reaction area of the patterned flow cell; (c) amplifying the DNA molecules on a reaction area of the patterned flow cell to form clusters; (d) contacting the individual DNA sequences in the DNA clusters with a set of probes with fluorescent dyes for each of the different target nucleic acid sequences of interest; (e) generating a signal in each reaction area containing the probes with fluorescent dyes; and (f) imaging the patterned flow cell.
 2. The method of claim 1, where the step of contacting the DNA molecules bind the suitable specific target probe set with attached fluorescent dye. 